Cholera is an epidemic diarrheal disease contracted by ingestion of the bacterium Vibrio cholerae. The WHO estimates that there are 3-5 million cases of cholera each year in the developing world resulting in approximately 100,000 deaths. While vaccines, rehydration therapy, and antibiotics are effective in the treatment of cholera, populations continue to be devastated because local resources are inadequate and the regional, national, and international responses too slow. Here we will investigate coordination of two carbohydrate-responsive signal transduction cascades that regulate V. cholerae metabolism, biofilm formation, and virulence. Our goal is to learn how to manipulate carbohydrate cues to shift V. cholerae to a low infectivity, low virulence state. Ultimately, we would like to use our findings to design readily available, inexpensive environmental additives and dietary modifications that decrease infectivity and virulence. The two signal transduction cascades we propose to study are known as the phosphoenolpyruvate phosphotransferase (PTS) and carbon storage regulatory (CSR) systems. Glucose-specific Enzyme llA (EllAGlc), a PTS intermediate and central regulator of carbohydrate transport and metabolism, imposes its control through direct interactions with other proteins. Here, we present the first evidence that EllAGlc interacts directly with MshH, a component of the CSR pathway, to accelerate degradation of the small regulatory csr RNA's. This activates virulence and biofilm formation. EllAGlc also interacts with adenylate cyclase (AC) to increase production of cyclic AMP. This results in repression of virulence and biofilm formation. Based on our preliminary data, we hypothesize that interaction of EllAGlc with the components of these opposing pathways is inversely regulated. In Aim 1, we will quantify the interaction of EllAGlc with AC and MshH under a variety of growth conditions. We will assess transcriptional and translational regulation of EllAGlc and its partners AC and MshH. In collaboration with Ethan Garner, we will use single molecule microscopy to study the redistribution of EllAGlc and its partners in the cell in real tim in response to environmental signals. We will also examine the structural basis of the interaction of EllAGlc with MshH. CsrA, an mRNA-binding protein, is the terminal component of the CSR pathway. We hypothesize that CsrA binds to mRNA targets that participate in V. cholerae metabolism, biofilm formation and virulence. In Aim 2, we will undertake genome-wide identification of CsrA targets by co-immunoprecipitation with CsrA and identification of precipitated RNAs by high throughput sequencing. These targets will be confirmed, and their role in biofilm formation and virulence will be investigated. We hypothesize that the CSR and PTS systems are required for mammalian disease. In Aim 3, we will use the neonatal rabbit model of cholera to assess the role of the PTS and CSR systems in intestinal colonization, virulence gene transcription, and elaboration of diarrhea.